Studies of Translation Initiation in Bacteria

基因表达的最后一步是翻译，即基于信使RNA （mRNA）序列的蛋白质合成。翻译是由核糖体完成的，核糖体是由核糖体RNA （rRNA）和蛋白质组成的亚细胞机器。近年来，x射线晶体学和低温电子显微镜揭示了核糖体的结构及其与各种底物和翻译因子的相互作用。尽管有如此丰富的结构信息，核糖体在翻译过程中如何促进特定事件的问题仍然不清楚。这个项目的目的是为了更好地理解翻译的起始阶段。起始是一个复杂的过程，需要从其他种类的tRNA中选择起始tRNA，并从mRNA中其他相似或相同的三联体序列中选择起始密码子。在细菌中，这一过程受到起始因子IF1、IF2和IF3的动力学控制。在其他生物体中，起始过程基本相似，但更为复杂，除了普遍保守的IF2 （eIF5B）和IF1 （eIF1A）外，还涉及10个或更多的因子。Fredrick实验室已经描述了小（30S）亚基rRNA中的许多突变，这些突变会导致起始缺陷。该项目将研究大（50S）亚基和因子IF1， IF3和S1的作用，进一步了解控制起始过程的分子机制。更广泛的影响：这个项目更广泛的影响在于它强调教育。该项目将提供(1)本科生和研究生的研究培训，(2)为当地高中生（哥伦布公立学校）提供暑期研究机会，(3)为俄亥俄州STEM（科学，技术，工程和数学）人才扩展计划的学生提供实验室实习，旨在提高STEM专业的成功率。此外，基因实验（与项目相关）将被纳入微生物学高级实验课程。通过进行实际的实验而不是实验室练习，这些学生将有机会在获得技术技能的同时进行发现。

The final step of gene expression is translation, the synthesis of protein based on the sequence of messenger RNA (mRNA). Translation is performed by ribosomes, sub-cellular machines composed of both ribosomal RNA (rRNA) and protein. In recent years, x-ray crystallography and cryo-electron microscopy have revealed the structure of the ribosome and its interactions with a variety of substrates and translation factors. Despite this wealth of structural information, questions of how the ribosome facilitates specific events during translation remain unclear. The goal of this project is to better understand the initiation phase of translation.Initiation is a complex process that requires selection of the initiator tRNA from other species of tRNA and selection of the start codon from other similar or identical triplet sequences in the mRNA. In bacteria, the process is kinetically controlled by initiation factors IF1, IF2 and IF3. In other organisms, initiation is fundamentally similar but more elaborate, involving 10 or more factors in addition to universally conserved IF2 (eIF5B) and IF1 (eIF1A). The Fredrick Lab has characterized a number of mutations in the rRNA of the small (30S) subunit that cause defects in initiation. This project will investigate the role of the large (50S) subunit and factors IF1, IF3, and S1, providing further insight into the molecular mechanisms that govern the initiation process. BROADER IMPACT: The broader impact of this project lies in its strong educational emphasis. This project will provide (1) research training for undergraduate and graduate students, (2) summer research opportunities for local high school students (Columbus Public Schools), and (3) laboratory internships for students of the Ohio STEM (Science, Technology, Engineering, and Mathematics) Talent Expansion Program, which aims to boost the success rate of STEM majors. In addition, genetic experiments (relevant to the project) will be incorporated into the curriculum of the advanced laboratory course in Microbiology. By conducting actual experiments rather than lab exercises, these students will be given the opportunity for discovery as they gain technical skills.